Protein-mediated facilitative glucose transport is essential for cellular metabolism, post-translation protein modification, cellular protection against oxidative stress and organismal carbohydrate homeostasis. In spite of extensive study, the mechanism of glucose transport is unknown. Our goal is to understand the molecular mechanism of protein-mediated glucose transport. To achieve this, we propose to investigate the structure and function of the human glucose transport protein GluT1. This transport protein is uniquely amenable to study being available as a purified protein from red cells, as a recombinant protein from Cos-7 cells and being amenable to function-screens by expression in glucose-transport null yeast. Specific aims 1 and 2 focus on structural determinants and functional consequences of GluT1 oligomerization in the cell membrane. Specific Aims 3 and 4 investigate transporter dynamics and catalysis. Specific Aim 1 tests the hypothesis that GluT1-specific sequence is required for GluT1 oligomerization. During the past grant cycle we discovered that cell membrane GluT1 is a homotetramer stabilized by GluT1-specific oligomerization sequence and preserved by some detergents but destabilized by others. We now challenge this hypothesis directly by GluT1 domain swapping and mutagenesis experiments asking whether transporter oligomeric structure and function are preserved. Specific Aim 2 tests the hypothesis that GluT1 oligomerization is necessary but not sufficient for glucose transport cooperativity. Some (but not all) GluT1 ligands promote cooperativity between GluT1 subunits. We have mapped ligand stereochemistry promoting cooperativity to the ligand but not yet to GluT1. We use constructs of specific aim 1 to ask whether GluT1 oligomerization is necessary for transporter cooperativity and examine GluT1 ligand binding site requirements for cooperativity in detail. Specific Aim 3 tests the hypothesis that specific GluT1 domains are conformationally dynamic and undergo accessibility changes in the presence of ligand by using mass spectrometry to map GluT1 domains exposed to solvent and to small, hydrophilic molecules that react with GluT1 in a substrate-dependent manner. Specific Aim 4 tests the hypothesis that the newly discovered "e(S)" - a transient, transport intermediate that excludes substrate from the aqueous environment - binds e1 and e2 ligands simultaneously. These biochemical and rapid quench-flow ligand binding studies critically challenge the tetramer and simple carrier models for transport and further investigate the nature of e(S) and the role of subunit cooperativity in substrate occlusion.